DNA AND CELL BIOLOGYVolume 21, Number 11, 2002© Mary Ann Liebert, Inc.Pp. 803–818

Defects in Pre-mRNA Processing as Causes of andPredisposition to Diseases

PETER STOILOV,1, p ERAN MESHORER,2,p MARIETA GENCHEVA,1 DAVID GLICK,2

HERMONA SOREQ,2 and STEFAN STAMM1

ABSTRACT

Humans possess a surprisingly low number of genes and intensively use pre-mRNA splicing to achieve thehigh molecular complexity needed to sustain normal body functions and facilitate responses to altered con-ditions. Because hundreds of thousands of proteins are generated by 25,000 to 40,000 genes, pre-mRNA pro-cessing events are highly important for the regulation of human gene expression. Both inherited and acquireddefects in pre-mRNA processing are increasingly recognized as causes of human diseases, and almost all pre-mRNA processing events are controlled by a combination of protein factors. This makes defects in these pro-cesses likely candidates for causes of diseases with complicated inheritance patterns that affect seemingly un-related functions. The elucidation of genetic mechanisms regulating pre-mRNA processing, combined withthe development of drugs targeted at consensus RNA sequences and/or corresponding proteins, can lead tonovel diagnostic and therapeutic approaches.

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OVERVIEW

RECENT PROGRESS IN THE HUMAN GENOME PROJECT has dem-onstrated that humans possess a surprisingly low number

of genes, estimated to range around 25,000 to 40,000 (Landeret al., 2001). A considerable fraction of the genes identified bydifferent approaches now appears to be nonoverlapping, whichimplies a somewhat larger number of genes than previously as-sumed; however, the total number is not drastically modified(Hogenesch et al., 2001). To create the proteome estimated torange between 90,000 and one million proteins (O’Donovan etal., 2001; Harrison et al., 2002; Hodges et al., 2002), humansabundantly process their pre-mRNAs (Modrek et al., 2001; Ru-bin, 2001) before protein translation occurs. This allows theproduction of multiple protein isoforms from a single gene.Transcription and pre-mRNA processing are both physicallyand functionally linked. This occurs by interaction of process-ing factors with the carboxy terminal domain of RNA poly-merase II (Steinmetz, 1997) and by interaction of processingfactors with transcription factors (Ge et al., 1998). As a result,the choice of a particular promoter influences splice site selec-

tion (Cramer et al., 1999, 2001). Finally, RNA processing fac-tors are linked to chromatin organizing elements by scaffold at-tachment factor B (Nayler et al., 1998). These associations im-ply that molecular defects in promoter structure andchromosomal localization of a gene can result in aberrant pre-mRNA processing.

While being transcribed, pre-mRNAs undergo a sequence ofstructural changes, such as capping, editing, splicing, andpolyadenylation. In this process, the fidelity of each maturationstep is controlled. Although the individual maturation steps canbe biochemically separated, recent reports show that most ofthem are functionally coupled (Bentley, 1999; Minvielle-Se-bastia and Keller, 1999; Maniatis and Reed, 2002). Defects inthis fine-tuned RNA assembly line are increasingly recognizedas causes of inherited human diseases (Philips and Cooper,2000; Stoss et al., 2000; Daoud et al., 2000; Dredge et al., 2001;Mendell and Dietz, 2001; Caceres and Kornblihtt, 2002; Nis-sim-Rafinia and Kerem, 2002). Changes in proteins function-ing at different steps during the processing or changes in theefficiency of the processes can result in variation of genetic ex-pression (Herbert and Rich, 1999). This may affect adaptation

1University of Erlangen-Nurenberg, Institute of Biochemistry, 91054 Erlangen, Germany.2The Hebrew University of Jerusalem, Institute of Life Sciences, Jerusalem 91904, Israel.p These authors contributed equally to the preparation of this manuscript.

events, when the expression of the genetic information is alteredaccording to changes in physiologic needs (Stamm, 2002). Itshould be pointed out that apart from pre-mRNA processing,which is a mechanism to enlarge the number of proteins madefrom the genome, there are a multitude of post-translational mod-ifications, such as phosphorylation, acetylation, glycosylation,cleavage, etc., which create functional diversity among existingprotein isoforms (O’Donovan et al., 2001; Hodges et al., 2002).

We propose that pre-mRNA processing has the potential toadapt the information stored in the genome to the physiologicrequirements of circumstance, place, and time. The failure ofsuch adaptation is frequently traced to defects in processing.These defects can manifest themselves directly in a disease ormay remain silent until some internal or environmental stimu-lus (e.g., stress) or time (i.e., old age) allows the mutation tobecome apparent. Furthermore, the sequential nature of pre-mRNA processing raises the interesting possibility thatpleiotropic diseases with a variable phenotype may be causedby specific compositions of trans-acting factors. For example,sporadic amyotrophic lateral sclerosis (ALS) was shown to beassociated with a change in splicing patterns of the glutamatetransporter gene EAAT2 (C. Lin et al., 1998), as well as changesin nitric oxide synthase (NOS) mRNA splicing (Catania et al.,2001). This indicates that the basic defect is in the pre-mRNAprocessing machinery, which gives rise to altered isoforms as-sociated with the disease. While the culprit gene(s) may not yetbe known, key proteins or RNAs controlling the affected pro-cesses can be subject to therapeutic efforts.

In this review, we will illustrate the variety of pathogenicdefects that occur at each step of pre-mRNA processing in eu-karyotic nuclei, and present examples for the mechanismsthrough which several mutations affect the pre-mRNA or theprocessing apparatus. To unravel the tangled interrelationshipsbetween these processes, we present examples of disease-caus-ing defects in each of the relevant stages of mammalian pre-mRNA processing, describe stress-induced changes in pre-mRNA processing as an example of a disease predisposition,and finally discuss potential prospects for the development ofnovel therapeutic approaches.

DEFECTS IN PRE-mRNA PROCESSING AS ADIRECT CAUSE OF HUMAN DISEASE

pre-mRNA editing

Nucleotides in the pre-mRNA can be chemically modifiedin a process called RNA editing (Gott and Emeson, 2000; Ger-ber and Keller, 2001; Bass, 2002). During editing, adenosineor cytosine is deaminated, changing it into inosine (translatedas guanine) or uridine, respectively. The editing of adenosinesis catalyzed by adenosine deaminases acting on RNA (ADARs)(Reenan, 2001), whereas the editing of cytosines is catalyzedby Apobec-1 (Chester et al., 2000). All of these enzymes aresubsequently associated with heterogeneous nuclear ribonucle-oprotein (hnRNP) complexes (Raitskin et al., 2001), and canaffect the removal of introns in the subsequent splicing reac-tion. For example, editing changes the splicing process inADAR2 by changing an AA into an AG at the 39 consensussplice site of the ADAR2 pre-mRNA, suggesting a close inter-

action between editing and splicing (Rueter et al., 1999). Thereare three ADAR genes in humans and genomic disruption ex-periments show that ADAR1 is essential in mice. The frequencyof editing a particular nucleotide is species, tissue, and devel-opment stage specific. In obese Zucker rats, editing of hepaticapolipoprotein B mRNA was found to be 42% higher than inlean controls, corresponding to 1.8-fold increase in Apobec-1catalytic activity in these rats (Phung et al., 1996). ADAR1 ex-pression was found to be upregulated in a mouse model of mi-crovascular lung injury (MLI) as well as in cultured alveolarmacrophages (MH-S cells) stimulated with endotoxin, or inter-feron-gamma, suggesting a plausible role for ADAR1 in thepathogenesis of MLI through induction by interferon (Rabi-novici et al., 2001). B cells with an unusual heavy- and light-chain antibody repertoire due to abnormal RNA editing wereobserved in patients suffering from rheumatoid arthritis, im-plying a role for RNA editing in autoimmune diseases (Meffreet al., 2000). Despite the ubiquitous expression of ADAR1 andADAR2, editing seems to be most prevalent in the brain, where1 out of 17,000 nt is edited (Paul and Bass, 1998). Assumingan average length of 1 kb for an mRNA, this implies that up to1 out of 20 brain transcripts is edited.

Perfectly matched RNA:RNA duplexes in the pre-mRNA ap-pear to be the major determinant for ADAR editing. Such du-plexes are created either by a natural regulatory “antisense” in-versely oriented region in the preedited transcripts or bytranscriptional readthrough of adjacent genes in antisense ori-entation. An example for the first mechanism involves Apobec-1, the editing of which requires a conserved 26 nt sequence thatcontains an 11 nt mooring sequence located 4–5 nt downstreamof the edited cytosine (Chester et al., 2000). Another examplerefers to malignant tumors, which edit the NF1 neurofibro-matosis transcripts more efficiently than benign tumors. Over-expressed Apobec-1, which catalyzes deamination of cytidines,induces murine tumorigenesis (Yamanaka et al., 1995), sug-gesting that the corresponding gene may operate as a proto-oncogene (Chester et al., 2000). Alternative splicing and RNAhyperediting was observed in the hematopoietic tyrosine phos-phatase (PTPN6) gene in CD341/CD1171 progenitor cells fromacute myeloid leukemia patients. There, editing of adenosine(7866) to guanine in a putative branch site causes retention ofintron 3, leading to abnormal accumulation of the aberrantsplice variants (Beghini et al., 2000). ADAR genomic disrup-tion in Drosophila causes no obvious developmental phenotypebut results in severe adult defects in motor control, flight, andmating. The defects increase in severity with age, concurrentwith neurodegeneration (Palladino et al., 2000; Reenan, 2001).Because editing is a prominent mechanism in the brain, im-pairments in this process were pursued in neurologic diseases.For example, Ca11 conductance of AMPA receptors is regu-lated by the GluR2 subunit that is edited to exchange a gluta-mine with an arginine residue. GluR2 editing efficiency andmRNA levels were significantly lower in the ventral gray areaof patients with ALS than in controls. These changes may ac-count for the enhanced Ca11 influx through AMPA receptors,which is a plausible cause for selective neuronal death in ALS(Takuma et al., 1999). Reduced editing efficiency of GluR2RNA was also observed in the prefrontal cortex of Alzheimer’sdisease patients and schizophrenics, as well as in the striatumof Huntington’s disease patients (Akbarian et al., 1995). This

STOILOV ET AL.804

points to neuronal activity as a contributing element of editingefficacy.

pre-mRNA splicing

Constitutive splicing. Almost all human genes contain intronsthat account for about 95% of the pre-mRNA (Lander et al.,2001). Higher eukaryotes possess several types of introns, whichhave slightly different mechanisms of excision splicing: a ma-jor one, in which the introns are flanked by GU-AG dinu-cleotides and which accounts for more than 98% of all introns(Lander et al., 2001), and a minor one in which introns areflanked by AU-AC dinucleotides (Tarn and Steitz, 1997). Themajor type has a subtype (about 1% of introns), for which theflanking nucleotides are GC-AG (Thanaraj and Clark, 2001).

During mRNA maturation those introns are excised and theremaining sequences, the exons, are joined. This process is per-formed by the spliceosome, a macromolecular complex com-prising five small ribonucleoprotein particles (snRNPs) and atleast 45 additional proteins (Neubauer et al., 1998).

The recognition of an exon is guided by three sequence el-ements of the pre-mRNA: the 59 and 39 splice sites and thebranchpoint. In addition, short sequences within or near theexon that can act as enhancers or silencers participate in exonrecognition. Both the 59 and 39 splice site sequences, the en-hancer/silencer elements, and the branch point associated withthe splicing process are short (8–12 nt) and degenerate. Never-theless, these sequences appear to be vulnerable to disease-caus-ing mutations that destroy splice sites or create novel ones. Fa-milial hypercholesterolemia is an autosomal dominant geneticlipoprotein disorder caused by defects within the low-densitylipoprotein receptor gene. Some of its many mutations wereshown to disrupt acceptor splice sites: an A to G substitutionin the penultimate 39-nucleotide of intron 16 (Lombardi et al.,1993) and a G to C transposition at the last nucleotide of in-tron 7 (Yu et al., 1999). In both cases a cryptic splice site wasactivated leading to the formation of a mutated receptor pro-tein. Another example of a splice site mutation that leads di-rectly to a disease phenotype is molybdenum cofactor defi-ciency (MoCoD), an inherited autosoml recessive disease thatleads to early childhood death. Two bi-cistronic genes, MOCS1and MOCS2, are responsible for the generation of the molybdo-enzymes, sulfite oxidase, xanthine dehydrogenase, and alde-hyde oxidase. A MOCS1 splice site mutation leads to the defi-ciency of all these molybdenum cofactor related enzymes (Reisset al., 1999). Other splice site mutations, including those in theCFTR and beta-globin genes, resulting in cystic fibrosis andthalassemias, were previously compiled (Krawczak et al., 1992;Nakai and Sakamoto, 1994). Mutations that alter the splicingprocess can occur outside both splice sites and enhancer/silencerelements. An example is the mutation described in a patientwith ataxia-telangiectasia (ATM) which represents a deletionin an intron-splicing processing element crucial for accurate in-tron removal (Pagani et al., 2002). The deletion of four nucle-otides in this element abolishes a binding site for U1 snRNPand leads to activation of a cryptic exon, thus producing an ab-normal mRNA transcript.

Changes in splicing factors can also be phenotypic. The ap-pearance of splicing-associated diseases is often correlated withthe plasticity and longevity of the affected cells. For example,

mutations in the human homologs of the splicing factor PRP31or the splicing factor PRPC8 may cause retinitis pigmentosa, aprogressive loss of rods and cones, which causes loss of over90% of vision during childhood (McKie et al., 2001; Vithanaet al., 2001).

Splice sites are highly degenerate, and additional regulatorysequences within the introns (intronic splicing enhancers) andthe exons (exonic splicing enhancers) have to be present forproper exon recognition (Hertel and Maniatis, 1998). These se-quences are also short and usually degenerate. They are boundby heterogeneous nuclear ribonucleoproteins, serine–arginine-rich (SR) proteins, and SR-like proteins. SR proteins are char-acterized by an RNA binding motif and an arginine–serine (i.e.,RS)-rich domain located at the carboxyl terminus (Tacke andManley, 1999; Graveley, 2000). This two-domain structure al-lows binding of SR proteins both to sequence elements on thepre-mRNA and to other components of the spliceosome. As aresult, multimolecular complexes that interact with pre-mRNAat multiple sites are formed, allowing the recognition and bring-ing together of distant sequences in the excision-splicing event(Dreyfuss et al., 2002).

SR protein binding sequences are frequently located in cod-ing exons; therefore, their degeneration and the degeneration ofthe genetic code allows the flexibility needed for compatibilitywith the coding requirements of the gene product. The bindingof SR proteins to their degenerate recognition sequences is in-trinsically weak, resulting in a concentration-dependent regu-lation, for example, certain sequence elements are recognizedonly at higher SR protein concentration (Manley and Tacke,1996). In addition to SR proteins, elements on the pre-mRNAbind to a diverse group of about 30 proteins, which are opera-tionally defined as components of hnRNP complexes.

hnRNPs contain RNA binding motifs as well as several aux-iliary domains, allowing those proteins to simultaneously bindto pre-mRNA and other proteins (Weighardt et al., 1996; Kre-cic and Swanson, 1999). Both SR proteins and hnRNPs arepresent in tissue characteristic concentrations (Kamma et al.,1995; Hanamura et al., 1998). SR proteins can be stored andreleased from cellular storage compartments as speckles,through phosphorylation (Misteli et al., 1998). As a result, theproper splice sites are recognized with a high degree of fidelityin an often tissue-specific manner.

An increasing number of alterations, both in normal andin alternative splicing, are being linked to defects in en-hancer/silencer sequences (Cooper and Mattox, 1997; Philipsand Cooper, 2000; Stoss et al., 2000; Cartegni et al., 2002).Several disease-relevant mutations are compiled in Table 1.As can be seen there, a number of these mutations are pres-ent in an exon, but do not change the protein sequence. Be-cause they cause aberrant splice site selection, they can causea disease, although they do not modify mRNA translation.Examples of these mutations include a T to C (L284L) mu-tation in tau exon 10 that disrupts an exonic splicing en-hancer, causing frontotemporal dementia with parkinsonismlinked to chromosome 17 (FTDP-17) (D’Souza et al., 1999),a C to G (R28R) mutation in porphobilinogen deaminase thatresults in exon 3 skipping, and which causes porphyria(Llewellyn et al., 1996) and a A to G mutation in pyruvatedehydrogenase resulting in Leigh’s encephalomyelopathy(De Meirleir et al., 1994).

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TABLE 1. MUTATIONS IN EXONIC REGULATORY SEQUENCES THAT CAUSE DISEASE

Gene/disorder Mutation Effect Reference

SMN2Spinal muscle atrophy

(SMA)

SMN1Spinal muscle atrophy

(SMA)

Beta-hexaminidaseBeta-subunit

Sandhof disease

TauFrontotemporal

Dementia withParkinsonism linkedto chromosome 17

Porphobilinogen deami-naseAcute intermittent por-

phyria

Integrin GPIIIaGlanzmann

thrombasthenia

FumarylacetoacetatehydrolaseHereditary tyrosinemia

type 1

Pyruvate dehydrogenaseE1 alphaLeigh’s

encephalomyelopathy

MNKMenkes disease

Adenosine deaminaseSevere combined immu-

nodeficiency disease

Silent C.T conversion inexon 7

425del5 W102X

Exon 11 P417L C.T conversion at nucleotide8

Intron 10 A.G conversionat position 217

Intron 10: 113 A.G, 114C.T, 116 C.T,IVS1013 G.A

Exon 10: L284L T.C,S305S T.C, S305NG.A

Exon 10: N297K T.G, del280K (AAG deletion)

R28R, C.G

C.A at position 116 andsilent G.A at 1134 ofexon 9

N232N C.T

Silent A.G

Gly.Arg G.A

R142X G.A and C.T inthe same codon

Disrupts ESE, skipping ofexon 7

Skipping of exon 3

Disrupts an ESE. Causesuse of a cryptic splicesite at nucleotide 1112

Causes usage of a crypticsplice site at position237. Disrupts ISE. Theconversion also disruptsa putative branch point

Disrupt ISEIVS1013 G.A improves

slightly the splice sitescore (from 6.8 to 7.0)

Disrupt ESES305N G.A improves the

splice siteDisrupts ESE

Skipping of exon 3

Skipping of exon 9

Skipping of exon 8

Aberrant splicing of exon 6

Skipping of exon 8

Skipping of exon 5

(Coovert et al., 1997;Jablonka et al., 2000;Lefebvre et al., 1995,1997; Lorson andAndrophy, 2000; Lorsonet al., 1999; Monani etal., 2000; Vitali et al.,1999)

(Sossi et al., 2001)

(Fujimaru et al., 1998)

(D’Souza et al., 1999;D’Souza andSchellenberg, 2000;Hasegawa et al., 1999;Hutton et al., 1998;Iijima et al., 1999;Spillantini et al., 1998;Stanford et al., 2000)

(Llewellyn et al., 1996)

(Jin et al., 1996)

(Ploos van Amstel et al.,1996)

(De Meirleir et al., 1994)

(Das et al., 1994)

(Santisteban et al., 1995)

Alternative splicing. In mammals, including humans mostgenes generate several mRNAs from a single gene by involve-ment of more than one pattern of excision-splicing choices, aprocess called alternative splicing (Graveley, 2001). For ex-ample, it has been estimated that 59% of all human genes onchromosome 22 are alternatively spliced. Because this numberis based on comparison of expressed sequence tags (ESTs) withgenomic data, the real number is likely to be higher, as lowabundant mRNAs are not taken into account, and ESTs are bi-ased towards the mRNA 39 ends (Lander et al., 2001). It islikely that the fine-tuned balance between SR proteins, hnRNPs,splice sites, and enhancer/silencer elements can be modulatedto achieve a change in pre-mRNA exon usage (Grabowski,

1998; Hastings and Krainer, 2001). The levels of SR proteinsand hnRNPs vary among tissues (Hanamura et al., 1998), andcan be further modulated by releasing SR proteins from in-tranuclear storage compartments, such as speckles, through pro-tein phosphorylation (Misteli et al., 1998). Alternative splicingis often tightly regulated in a cell type- and/or development-specific manner; cells from the immune and the nervous sys-tems use this mechanism most abundantly (Grabowski andBlack, 2001; Lander et al., 2001).

The phenotype of most neurodegenerative diseases manifestsitself later in life, and some missplicing events are causally re-lated to such diseases. Mutations in tau exon 10 that are im-plicated in FTDP-17, are good examples of such a complex phe-

DEFECTS IN PRE-mRNA PROCESSING 807

TABLE 1. MUTATIONS IN EXONIC REGULATORY SEQUENCES THAT CAUSE DISEASE (CONT’D)

Gene/disorder Mutation Effect Reference

The affected gene (bold) and the disease is listed in the first column, the mutation in the second column. The effect on pre-mRNa splicing and the assumed mechanism are listed in the third column.

Arylsulfatase AMetachromatic leuko-

dystrophy

Fibrillin-1Marfan syndrome

CYP 27Cerebrotenidinous xan-

thomatosis

CD45Individuals do not suffer

from obvious immu-nodeficiency, but it isnotable that nohomozygotes havebeen described.Associated withMultiple sclerosis inthe American popula-tion.

Beta-globinBeta-Thalassemia

BRCA1Breast and ovarian cancer

NF-1Neurofibromatosis type 1

DMPKMyotonic dystrophy

Thr409Ile C.T

Silent C.T in exon 51

Silent G112G G.T in exon 2

Silent C.G at position 77of the alternative exon 4

T-.G transversion in posi-tion 705 of intron 2

E1694X G.T

Y2264X C.A, C.GR304X C.TQ756X C.T

Expanded (CUG).40 inthe 39 UTR of DMPK

Activates a cryptic splicesite

Skipping of exon 51

Creates a cryptic splicesite. In addition it causesskipping of the entireexon 2

Disrupts an ESE.Constitutive inclusion ofexon 4

Activates cryptic 39 splice site in intron 2

Disrupts ESE and causesskipping of exon 18

Skipping of exon 37Skipping of exon 7Skipping of exon 14

Two possibilities: CUGrepeats sequestrateCUG-BP and thereforeprevent it from its nor-mal function; or expand-ed CUG repeats alter thealternative splicing ofthe DMPK-mRNA bydeveloping a new 39splice site.

(Hasegawa et al., 1994)

(Liu et al., 1997)

(Chen et al., 1998)

(Jacobsen et al., 2000;Lynch and Weiss, 2001;Zilch et al., 1998)

(Dobkin and Bank, 1983;Dobkin et al., 1983)

(Liu et al., 2001)

(Ars et al., 2000a, 2000b;Hoffmeyer et al., 1998;Messiaen et al., 1997)

(Phillips et al., 1998;Tiscornia andMahadevan, 2000)

notype. Frontotemporal dementias represent a rare form of pre-senile dementia, and are clinically defined by behavioral andpersonality changes, psychomotor stereotypes, as well as lossof judgment and insight. The neuropathologic findings includeasymmetric frontotemporal atrophy and the presence of fila-mentous tau deposits. FTDP-17 was mapped to the tau locuson chromosome 17, and at least 50 kindreds are affected worldwide (Hutton et al., 1998). Tau transcripts undergo complexregulated splicing in the mammalian nervous system. The al-ternative splicing of tau’s exon 10 is species specific: this exonis excised in adult humans, but in the adult rodent brain, it isused constitutively. Exon 10 encodes one of the four micro-tubule binding sites of tau. In the normal brain, tau isoformscontaining and lacking exon 10 sequences are balanced. A dis-ruption of the proper distribution of tau isoforms is observed inthe pathology of several tauopathies. In FTDP-17, sporadic cor-ticobasal degeneration and sporadic progressive supranuclearpalsy, tau proteins containing exon 10 sequences are overpro-duced, whereas in sporadic Pick’s disease tau proteins lackingexon 10 sequences are predominant (Goedert et al., 1999;Spillantini and Goedert, 2001).

Mapping of elements in tau exon 10 has revealed a poten-tial stem-loop structure at the 59 splice site, which may be in-volved in regulation of exon 10 splicing (Hutton et al., 1998).In addition, a complicated set of exonic enhancer elements wasidentified (D’Souza and Schellenberg, 2000; Gao et al., 2000).The disruption of these elements by mutation apparently dis-turbs the balance between the different tau isoforms and causesthe aggregation of improperly spliced gene products associatedwith neurodegeneration.

Misdirected regulation of alternative splicing is observed inseveral additional neurologic disorders. One example includesschizophrenia. The alternative splicing of the long (L) and short(S) gamma2 subunit mRNAs of the gamma-amino butyrate typeA (GABA-A) receptor was shown to be modified in the pre-frontal cortex of schizophrenic patients. The reduction ingamma2S and the increase in gamma2L mRNAs were sug-gested to result in less active GABA-A receptors with severeconsequences for cortical integrative function (Huntsman et al.,1998). Postmortem brain investigations revealed alternativesplicing of N-methyl-D-aspartate (NMDA) R1 (NRI) carboxy-terminus isoforms in the superior temporal gyrus of schizo-phrenic patients (Le Corre et al., 2000). Schizophrenia was fur-ther shown to be associated with alternative splicing of theneural cell adhesion molecule (N-CAM) mRNAs. Elevated lev-els of the variable alternative spliced exon (VASE) were foundin CSF of schizophrenic patients (Vawter et al., 2000).

39End processing and nuclear export

Once RNA polymerase II encounters the highly conservedAAUAAA polyadenylation signal, 39 end processing takesplace (Dreyfuss et al., 2002). A minimum of six factors are re-quired for 39 end formation: cleavage and polyadenylation fac-tors (CPSF), cleavage stimulation factor (CSF), cleavage fac-tors I and II, poly(A) polymerase (PAP), and poly(A) bindingprotein. These factors recognize the AAUAAA signal and adownstream element and then perform cleavage of the RNA atits 39 end as well as its polyadenylation.

Thalassemia is one of the world’s most common hereditary

diseases. Common forms of both alpha- and beta-thalassemiaare both associated with point mutations within the polyadeny-lation signals of alpha-globin and beta-globin genes, respec-tively (Higgs et al., 1983; Orkin et al., 1985), leading to thegeneration of abnormal hemoglobin. Patients suffer from mod-erate anemia with microcytosis and hypochromia.

A guanosine to adenosine mutation in the 39 end of the pro-thrombin gene is a common cause for thromboembolic events.This mutation is positioned at the site where the pre-mRNA iscleaved during processing and then polyadenylated. The muta-tion leads to more efficient cleavage of the pre-mRNA, and con-sequently, to elevated amounts of prothrombin mRNA and anincrease of the blood prothrombin concentration (Gehring et al.,2001). The mutation is common, with an allele frequency of1.2%, and may represent an advantage in young age by pro-viding better blood clotting, for example, during childbirth(Poort et al., 1996). Another example for misregulation of 39end formation is autosomal dominant oculopharyngeal muscu-lar dystrophy (OPMD), an adult-onset disease caused by a(GCG)8–13 repeat expansion in the polyadenylation bindingprotein 2 (PABP2) gene. Patients suffer from progressive dys-phagia, eyelid ptosis, and proximal limb weakness, and itspathologic hallmark is the accumulation of intranuclear inclu-sions in muscle fibers due to abnormal mRNA processing (Braiset al., 1998). The export of mRNAs into the cytosol is medi-ated by nuclear export factors (NXFs) believed to be the mo-lecular link between the hnRNP complexes and the nuclear porecomplex (Herold et al., 2000). Loss of the NXF5 protein, whichis most likely a novel nuclear RNA export factor, results in X-linked mental retardation, but the molecular details remain tobe worked out (J. Lin et al., 2001).

Nonsense mutations, exon skipping, and mRNAdegradation

Premature stop codons may lead to an alternatively splicedvariant that skips the mutated exon (Dietz et al., 1993), a phe-nomenon referred to as nonsense-mediated altered splicing.Nonsense-mediated altered splicing was thought to require nu-clear recognition by the spliceosome of “cytosolic” translationalsignals but recent research on the BRCA1 gene suggests it canresult from disruption of a splicing enhancer located in the cod-ing sequence (H.X. Liu et al., 2001). A related mechanism isnonsense mediated decay (NMD), also known as RNA sur-veillance. In both cases, the nascent mRNA is screened for non-sense mutations. NMD takes place if the distance between astop codon and the downstream exon–exon junction is largerthan 50–55 nt (Hentze and Kulozik, 1999; Maquat andCarmichael, 2001). Such stop codons are considered premature,and NMD leads to decapping and degradation of the mRNA.Because about 25% of all alternatively spliced exons introducealternative stop codons (Stamm et al., 2000), NMD togetherwith alternative splicing is a common mechanism to downreg-ulate gene expression. The location of exon–exon junctions ismemorized in the cytosol by binding of the Y14 protein to thejunction (Kim et al., 2001; Kim and Dreyfus, 2001). Messen-ger RNA stability is also decreased by adenylate, uridine-richinstability elements (ARE) that accelerate mRNA deadenyla-tion. AREs are usually located in the 39UTR and bind to hn-RNPs of the HuR/HuA family (Brennan and Steitz, 2001).

STOILOV ET AL.808

Chain termination mutations are frequent causes for disease inhumans. For example, 89% of mutations in the ATM gene thatcauses ataxia-telangiectasia and 77% of mutations in BRCA1give rise to premature stop codons (Couch and Weber, 1996;Gilad et al., 1996). The mRNAs with premature stop codonsrarely produce truncated proteins but are rather subject to non-sense-mediated decay. Typically, these mutations result in aloss-of-function phenotype. The involvement of NMD in beta-thalassemia, gyrate dystrophy, and Marfan syndrome has beenrecently reviewed (Frischmeyer and Dietz, 1999). A change inRNA stability is often associated with cancer, due to stabiliza-tion of mRNAs that promote tumor progression, such as im-munosuppressive cytokines and angionogenic growth factors.Interestingly, HuR, a factor that stabilizes mRNAs by bindingto AREs is constitutively expressed in brain tumors, and its ex-pression pattern correlates with the grade of malignancy(Nabors et al., 2001).

DEFECTS IN PRE-mRNA PROCESSING THAT PREDISPOSE TO A DISEASE

Stress-induced alterations

A number of mutations do not show a phenotype directly,but rather remain silent, until some external stimulus allows themutation to become apparent in certain cell types or develop-mental and aging phases. This implies that diagnostic and ther-apeutic approaches might be developed to anticipate, and pre-vent the damage caused by such defects. The consequences ofstress present an example of such a circumstance.

In recent years, a growing body of evidence links alternativesplicing adaptation to stressful internal and external changes incells and tissues. The term stress, adapted from physics, spansphysical, chemical, and psychologic pressures that are poten-tially detrimental and sharing common features (McEwen,1999). The cellular signaling pathways affecting alternativesplicing under modified stimuli are poorly understood, althoughrecent efforts by several investigators yielded interesting find-ings. Activation of the MKK3/6-p38 cascade was shown to mod-ify the subcellular distribution of hnRNP A1, resulting in mod-ified alternative splicing (van der Houven van Oordt et al.,2000). The ability of hnRNP K to silence mRNA translationalso requires relocalization to the cytoplasm, which is depen-dent on the stress-activated ERK MAP–kinase pathway (Ha-belhah et al., 2001). The ERK/MAP–kinase pathway wasshown to be involved, upon activation of T cell lymphocytes,in the alternative splicing of CD44 by retaining exon v5 se-quence in the mature mRNA (Weg-Remers et al., 2001). In-triguingly, some of the evoked changes in alternative splicingproduce transcripts that have antagonistic cellular actions. Forexample, heat-shock factor 4 generates by alternative splicingboth an activator and a repressor of downstream heat-shockgenes (Tanabe et al., 1999). Also, alternative splicing of theapoptotic gene, bcl-x, substitutes a large protein product, Bcl-xL, which inhibits cell death, for a smaller one, Bcl-xS, whichantagonizes this ability under certain conditions (Boise et al.,1993). Neuronal activity-dependent alternative splicing wasshown for the rat homolog of the human splicing regulator trans-former 2 gene, hTra2-beta, in the rat brain (Daoud et al., 1999).

Stress-induced changes in alternative splicing have beendemonstrated for the transcripts of a variety of genes, includ-ing those for heat-shock factors 1 (Goodson et al., 1995) and4 (Tanabe et al., 1999), the apoptotic gene bcl-x (Boise et al.,1993), genes for several Jun N-terminal kinases (JNKs) in-volved in regulating transcription factors (Holland et al., 1997;Y. Zhang et al., 1998) and the human genes for the ATF fam-ily of transcription factors (Goetz et al., 1996). A change in al-ternative splicing following stress was demonstrated, for example, for the glucocorticoid receptor (GR). Perinatal ma-nipulations and postnatal handling were shown to selectivelyelevate GR mRNA containing a hippocampus-specific exon,which facilitates adaptation to the conditions of stress. Prena-tal glucocorticoid exposure, in turn, increased hepatic GR ex-pression by producing a minor exon 1 variant (McCormick etal., 2000).

Glucocorticoids also regulate the splicing pattern of themurine Slo gene encoding a brain K1-channel, which dependson neuronal depolarization (Xie and McCobb, 1998). Hypoxiawas shown to induce the alternative splicing of the presenilin-2 gene, generating an isoform also found extensively in thebrain of Alzheimer’s disease patients (Sato et al., 1999). A de-tailed study of SR proteins under ischemic conditions in thebrain revealed that some of them translocate from the nucleusto the cytosol after an ischemic event, which could explain theobserved changes in splice site selection (Daoud et al., 2002).Although splice site selection can serve as a physiologic adap-tation to a change in the external conditions, the above exam-ples show that these changes may also contribute to patho-physiologic events. One extensively studied example is achange in the acetylcholinesterase (AChE) pre-mRNA pro-cessing (Soreq and Seidman, 2001). Here, various external stim-uli were shown to induce rapid, long-lasting changes of neu-ronal AChE pre-mRNA splicing. These include psychologicstress (Kaufer et al., 1998), environmental stimuli (anti-AChEintoxication) (Shapira et al., 2000), head injury (Shohami et al.,2000), and inherited AChE overexpression (Meshorer et al.,2002). Although most of the currently available evidence onthis splicing shift refers to animal studies, a recent screen dem-onstrated accumulation of the stress-induced AChE-R variantin the cerebrospinal fluid (CSF) of patients with stress indices,indicating that these pathologic changes also occur in humans(Tomkins et al., 2001).

Tumorigenesis

Many cases of different cancers are also associated withchanges in the splicing pattern of various genes. For example,43% of neurofibromatosis type 1 (NF1) defects are caused byaberrantly spliced pre-mRNA (Ars et al., 2000b). Furthermore,the splicing pattern of the adhesion molecule CD44 changesduring tumorigenesis (Stickeler et al., 1999) and melanomaswere found to be associated with elevated levels of the delta1band deta1d alternatively spliced transcripts of the tyrosinasegene compared to normal skin melanocytes (LeFur et al., 1997).Different splice variants of the estrogen receptor (ER) (e.g.,skipping of exons 2, 3, 4, 5, or 7 (Q.X. Zhang et al., 1996))were found to be associated with human breast cancer. Resis-tant acute myeloid leukemia was shown to be associated withhigh incidence of alternatively spliced forms of deoxycytidine

DEFECTS IN PRE-mRNA PROCESSING 809

kinase, which could contribute to the process of cytarabine re-sistance in these patients (Veuger et al., 2000). It was shownthat SR proteins display tumor stage-specific changes in mam-mary tumorigenesis (Stickeler et al., 1999), which suggests thatconcentration alterations of these regulatory proteins could or-chestrate the use of different splice sites in tumors.

Pathologies associated with changes in alternative splicingpatterns without specific mutations in splice sites may reflectdefects in an upstream splicing modifier. Differential expres-sion of hnRNPs, snRNPs, SR, and SR-like proteins could, inprinciple, be the cause for such pathologies. For example, po-sitional cloning revealed segregation with prostate cancer ofELAC2, which displays sequence similarity to the 73-kD a sub-unit of mRNA 39 end cleavage and polyadenylation specificityfactor (CPSF73) (Tavtigian et al., 2001), suggesting mRNAprocessing abnormalities in prostate cancer.

OUTLOOK FOR THERAPY

A general theme in pre-mRNA processing is a high fidelityand specificity achieved by combinatorial control. This is ex-emplified in the redundancy of the relevant sequences, the mod-ular composition of the corresponding proteins, and the cascadepattern of the involved processes. The examples compiledabove show that numerous defects in pre-mRNA processingcontribute to human diseases. This, in turn, calls for the devel-opment of diagnostic and therapeutic means targeted at pre-mRNA processing. Different approaches have been tested invarious models of diseases that are summarised in Table 2.

Antisense oligonucleotides

The danger of nonspecificity in targeting pre-mRNA pro-cessing can be avoided by design of selective agents, such asoligonucleotides (Mercatante and Kole, 2000) (Fig. 1A). Beta-thalassemia in human erythroid cells may be treated with mod-ified oligonucleotides binding to mutated splice sites, showingthat this approach is feasible (Lacerra et al., 2000). Antisenseoligoribonucleotides targeted against an aberrant 59 splice sitewere also used to partially reverse the aberrant splicing of beta-globin mRNA in beta-thalassemia/HbE disease cells in culture(Shirohzu et al., 2000). In cultured cells, targeting of splice sitesby antisense oligonucleotides was shown to reverse the inclu-sion of tau exon 10 in a model of FTDP-17 (Kalbfuss et al.,2001) and to increase the inclusion of SMN exon 7, associatedwith spinal muscular atrophy (Lim and Hertel, 2001). 29-O-Methylated antisense oligoribonucleotides were used to mod-ify the splicing pattern of the dystrophin pre-mRNA in the mdxmouse model of Duchenne muscular dystrophy (DMD). DMDis a severe muscle disease caused by defects within the dys-trophin gene. Its milder version, Becker muscular dystrophy, iscaused by in-frame deletions that generate a shorter but mini-mally functional dystrophin protein. This suggested removal ofthe deficient part in the mutated dystrophin as a therapeutic ap-proach. The oligoribonucleotides blocked binding sites in-volved in normal dystrophin pre-mRNA splicing, inducing ex-cision of exon 23 with the mdx nonsense mutation, withoutdisrupting the reading frame (Mann et al., 2001).

Finally, the detrimental accumulation of the stress-inducedAChE-R mRNA can be prevented by antisense oligonucleotides,

STOILOV ET AL.810

TABLE 2. OVERVIEW OF THE THERAPEUTIC STRATEGIES

Drug/substance Gene/disease tested Reference

Antisense oligonucleotidesModified oligonucleotides b-thalassemia (Shirohzu et al., 2000)

FTDP-17 (Kalbfuss et al., 2001)SMN2/Spinal muscular (Lim and Hertel, 2001)

athrophyDystrophin/Duchenne (Mann et al., 2001)

muscular dystrophyAChE-R (Shohami et al., 2000; Lev-Lehman et al., 2000)

RNAiRNA oligonucleotide Multiple (Cellotto and Graveley, 2002)

RibozymesRibozyme Mutant b-globin (Lan et al., 1998)

p53 mRNA trans-splicing (Watanabe and Sullenger, 2000)SMaRT

Targeting RNA CFTR exon 10 (Mansfield et al., 2000)Low molecular weight drugs

Neomycin FTDP-17 (Varani et al., 2000)Aclarubicin SMN2/spinal muscular (Andreassi et al., 2001)

athrophySodium butyrate SMN2/spinal muscular (Chang et al., 2001)

athrophyExpression of trans-acting factors

tra2-b1 SMN2/spinal muscular (Hofmann et al., 2000)athrophy

clk-1 tau/FTDP-17 (Hartmann et al., 2001)

The principal action is shown in a gray box. Drugs and substances used are in the first column, genes and diseases tested arelisted in the second column.

with potentially promising prospects for the treatment of headinjury (Shohami et al., 2000) and exposure to organophosphateacetylcholinesterase inhibitors (Lev-Lehman et al., 2000).

RNAi

The most recent and yet mechanistically illusive phenome-non that may lead to future therapeutic technologies is RNA in-terference (RNAi). Found by serendipity, RNAi acts to causetarget-specific gene silencing by destabilizing cellular RNA(Carthew, 2001; Zamore, 2001). Intriguingly, RNAi was dem-onstrated to be effective in vivo in Caenorhadditis elegans whenthe nematodes digested engineered bacteria that expressed dou-ble-stranded RNA of the C. elegans unc-22 gene. These ne-matodes developed similar phenotypes of unc-22 mutants (Timmons and Fire, 1998). RNAi was shown to suppress gene-expression levels in mammalian cells using either short (e.g.,22-mer) double-stranded RNAs (Elbashir et al., 2001), or long(e.g., 500-mer) dsRNAs (Paddison et al., 2002), as well as sta-ble “knock-down” of genes using long hairpin dsRNA (Paddi-son et al., 2002). Recently, RNAi was used to selectively de-grade alternatively spliced mRNA isoforms in Drosophila bytreating cultured cells with dsRNA corresponding to an alter-natively spliced exon (Celotto and Graveley, 2002). This pow-erful tool may come to occupy an important role in future genetherapy (Fig. 1B).

Ribozymes

Ribozymes are RNAs that catalyze a limited number of re-actions in cells, especially cleavage of other nucleic acids(Lewin and Hauswirth, 2001). The ribozymes under develop-ment for therapy are based on small naturally occurring RNAs,such as the hammerhead and the hairpin, derived from plantvirus satellite RNA, the tRNA processing ribonuclease P(RNase P), and group I and group II ribozymes, which occuras introns in organelles and bacteria but can be engineered toact in trans on RNA or DNA. Group I introns have been usedto repair defective mRNAs by trans-splicing, for example, toreplace defective p53 mRNA with the wild type, resulting inclose to full restoration of the wt p53 activity (Watanabe andSullenger, 2000). A trans-splicing group I ribozyme was alsoshown to be a useful candidate for altering the mutant beta-glo-bin transcripts in erythrocyte precursors derived from periph-eral blood of sickle cell disease subjects. Sickling beta-globin,the beta-chain of HbS, transcripts were converted into messen-ger RNAs that encode the nonsickling beta-globin (Lan et al.,1998).

SMaRT

Another novel approach for gene therapy is Spliceosome-Mediated RNA Trans-splicing (SMaRT) (Puttaraju et al.,1999). SMaRT is an emerging technology in which RNA mol-ecules are designed to code a specific pre-mRNA by utilizingtrans-splicing reaction between the introduced RNA and its pre-mRNA target (Fig. 1C). During the splicing reaction, a part ofthe pre-mRNA is first excised in a concerted reaction, followedby the remaining exons being ligated. This reaction occurs nor-mally in cis, for example, on a single pre-mRNA molecule that

is transiently attached to the spliceosome. However, splicingcan also take place between two independently transcribed se-quences, a process called trans-splicing that is found in try-panosomes, nematodes, flatworms, and plant mitochondria. Be-cause the mechanisms of trans- and cis-splicing are similar, itis possible to generate RNA molecules that would be processedby the spliceosome in a trans-splicing reaction, which can beused to repair a defective pre-mRNA molecule by exchangingparts of it (Puttaraju et al., 1999).

SMaRT was demonstrated successfully using plasmids ex-pressing mutant CFTR minigenes. When 293T cells were co-transfected with both the mutated and the normal constructs,they produced a trans-spliced mRNA in which the mutant exon10 was replaced by a normal one. This trans-splicing reactionwas further shown to produce mature CFTR protein (Mansfieldet al., 2000).

Low molecular weight drugs

The use of RNA-biding antibiotics such as gentamicin, chlo-ramphenicol, and tetracycline clearly shows that RNA and/orRNA interacting proteins can be targeted by drugs (Varani etal., 2000; Xavier et al., 2000). Targeting pre-mRNA process-ing pathways could therefore be a new therapeutic approach.Aclarubicin, an anticancer drug, was shown to change the al-ternative splicing pattern of SMN2 in cells derived from spinalmuscular atrophy patients. In this disease, the SMN1 gene isdeleted, and an almost identical human gene, SMN2, cannotcompensate for the loss, because it is differently spliced.Aclarubicin treatment can reverse this wrong splicing pattern,which results in formation of full length SMN protein. Althoughit is yet unknown how aclarubicin changes alternative splicing(Andreassi et al., 2001), this is a promising example of pre-mRNA therapeutics (Fig. 1D).

Indirect effects

The combinatorial regulation of pre-mRNA processing mayalso result in indirect effects that have to be considered for anytherapeutic approach. This is illustrated by the change in alter-native splicing patterns of human cardiac troponin T in patientswith myotonic distrophy. Myotonic dystrophy is caused by aCUG repeat extension in the 39 UTR of the cAMP-dependentprotein kinase gene (Korade-Mirnics et al., 1998). This repeatextension causes a sequestration of a CUG-binding protein,which results in a change of cardiac troponin T pre-mRNAsplicing patterns (Philips et al., 1998; Lu et al., 1999). Increasedexpression of the CUG-binding protein in skeletal muscle tis-sue of myotonic dystrophy patients further results in aberrantalternative splicing of the insulin receptor pre-mRNA, with pre-dominant expression of the nonmuscle isoform (Savkur et al.,2001). Finally, the splicing pattern of mutated alleles, for ex-ample in the cystic fibrosis CFTR gene, strongly depends onthe cell type (Rave-Harel et al., 1997), explaining why naturalmutations frequently have a tissue- or cell type-specific effect[e.g., male sterility due to impaired testicular splicing of theCFTR transcripts (Kerem and Kerem, 1996]). A possible ex-planation would be a cell type-specific set of regulatory fac-tors. Skipping of CFTR exon 9 was shown to depend on ex-pression of a factor binding a polymorphic repeated sequencein its 39 splice site, TDP-43 (Buratti et al., 2001). However, this

DEFECTS IN PRE-mRNA PROCESSING 811

STOILOV ET AL.812

FIG. 1. Principles of different therapeutic strategies. Exons are indicated as boxes, introns as thick lines. Different coloring in-dicates alternative exon usage. Splicing patterns are shown as thin lines. Small molecular weight substances are shown as smallyellow polygons. Base pairing is indicated by thin lines. (A) Antisense oligonucleotides (thick red line) can bind to a splice siteand prevent its usage (left), which favors exon skipping (right). (B) Misspliced mRNAs can be eliminated by RNAi using spe-cific siRNAs (green lines). As a result, the undesired splice product is eliminated (right). (C) Undesired exons (red) can be re-placed using SMaRT (yellow) constructs, which results in the exchange of an exon in a trans-splicing reaction (right). (D) Smallmolecular weight molecules (yellow) can change splicing patterns, for example, by stabilization of secondary pre-mRNA struc-tures. (E) Phosphorylation of splicing regulatory proteins changes their interaction with RNA or other proteins and can result inan altered splicing product in the presence (top) or absence (bottom) of a kinase activity (black enzyme).

effect also depends on the simultaneous expression of SR pro-teins. It is clear that this combinatorial control will impact onevery therapeutic intervention; therefore, the effects of existingdrugs on pre-mRNA processing of yet unknown gene products,in addition to their target molecules, should be taken into con-sideration. Nevertheless, the importance of such processespoints to the RNA transcripts themselves, as well as their cog-nate proteins, as novel targets for therapeutic intervention.

Trans-Acting factors

Spinal muscular atrophy illustrates how the knowledge ofthe molecular mechanisms regulating pre-mRNA processing al-lows the development of rational therapies. The disease iscaused by the loss of the Survival of Motor Neuron gene 1(SMN1) and subsequent loss of the SMN protein. As notedabove, a nearly identical gene, SMN2, gives rise predominantlyto a shorter mRNA form, producing a modified SMN protein.Therefore, SMN2 cannot compensate for the absence of SMNprotein. The loss of the SMN1 gene can, however, be compen-sated in cultured cells by manipulating the splicing pattern ofSMN2, through increasing the amount of a regulatory factor,tra2-beta1 that binds to a purine-rich enhancer of the alterna-tive exon (Hofmann et al., 2000). Several SR proteins bind tothis enhancer, and their ratio can also be changed by sodiumbutyrate. Administration of this drug to lymphocytes of spinalmuscular atrophy patients results in an altered splicing patternof SMN2, which could also compensate for the loss of SMN1(Chang et al., 2001).

In FTDP-17 mutations, splicing of tau exon 10 is mis-regu-lated through complex changes in the composition of severalsplicing enhancers (D’Souza and Schellenberg, 2000). Thesemutations can be overcome by expressing cdc2 like kinases(clk1-4) (Nayler et al., 1997). These kinases phosphorylate SRproteins and alter tau exon 10 splicing, most likely by chang-ing the phosphorylation-dependent composition of the enhancercomplex (Hartmann et al., 2001; Fig. 1E). Low molecularweight drugs activators of clk1-4 kinases, when identified, maybe therapeutically beneficial for this devastating disease.

ACKNOWLEDGMENTS

This work was supported by the Deutsche Forschungsge-meinschaft (SFB473, Sta399/5-1, Sto456/1-1 and Sta399/7-1,to S.S.) and the U.S. Army Medical Research and MaterielCommand (DAMD 17-99-1-9547, to H.S.).

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Address reprint requests to:Stefan Stamm, Ph.D.

University of Erlangen-NurenbergInstitute of Biochemistry

Fahrstrasse 1791054 Erlangen, Germany

E-mail: stefan@stamms-lab.net

Received for publication April 9, 2002; received in revised formJuly 20, 2002; accepted July 31, 2002.

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